Enhanced fungal specificity and in vivo therapeutic efficacy of a C-22-modified FK520 analog against C. neoformans.

IMPORTANCE: Fungal infections cause significant morbidity and mortality globally. The therapeutic armamentarium against these infections is limited, and the development of antifungal drugs has been hindered by the evolutionary conservation between fungi and the human host. With rising resistance to the current antifungal arsenal and an increasing at-risk population, there is an urgent need for the development of new antifungal compounds. The FK520 analogs described in this study display potent antifungal activity as a novel class of antifungals centered on modifying an existing orally active FDA-approved therapy. This research advances the development of much-needed newer antifungal treatment options with novel mechanisms of action.

Scanning Quadrupole Data-Independent Acquisition, Part B: Application to the Analysis of the Calcineurin-Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs.

Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded an assessment of the precision of the method as demonstrated by peptide- and protein-centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically relevant antifungal drugs that are guideline recommended for the treatment of invasive "aspergillosis" caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin), which specifically target the fungal plasma membrane and the fungal cell wall, respectively, were chosen to distinguish variations occurring in the proteins coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell-wall biosynthesis and septation. Furthermore, abundant coimmunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment, indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurin-dependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies.

Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach.

Increases in the incidence and mortality due to the major invasive fungal infections such as aspergillosis, candidiasis and cryptococcosis caused by the species of Aspergillus, Candida and Cryptococcus, are a growing threat to the immunosuppressed patient population. In addition to the limited armamentarium of the current classes of antifungal agents available (pyrimidine analogs, polyenes, azoles, and echinocandins), their toxicity, efficacy and the emergence of resistance are major bottlenecks limiting successful patient outcomes. Although these drugs target distinct fungal pathways, there is an urgent need to develop new antifungals that are more efficacious, fungal-specific, with reduced or no toxicity and simultaneously do not induce resistance. Here we review several lines of evidence which indicate that the calcineurin signaling pathway, a target of the immunosuppressive drugs FK506 and cyclosporine A, orchestrates growth, virulence and drug resistance in a variety of fungal pathogens and can be exploited for novel antifungal drug development.

Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus.

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.